Single molecule sequencing (SMS) methods, such as nanopore sequencing, have certain advantages over other next generation sequencing approaches. In particular, SMS is rapid and produces long read lengths. However, conventional SMS methods are characterized by a high error rate in raw reads. Error rate can be presented as a % error, corresponding to the number of errors per 100 called bases. Alternatively, error rate can be presented as a “Q” value. “Q,” can be calculated using the following formula: −10×log10(P), where P is the probability of an incorrect base call. See, Ewing & Green, 1998, Genome Res. 8:186-194. For example, Q10 refers to a 1 in 10 probability of an error, or a 90% accuracy, and Q30 refers to a 1 in 1000 probability of an error, or a 99.9% accuracy. Nanopore sequencing has been reported to provide a base-call accuracy in the range of only Q5 to Q7 (about 70-85%). Other SMS methods (e.g., zero-mode waveguide sequencing; SMRT Pacific Biosciences) also suffer from high error rates.
Nanopores and methods of sequencing using nanopores are known in the art. See, e.g., Clarke et al., 2009, “Continuous base identification for single-molecule nanopore DNA sequencing,” Nature Nanotechnology 4:265-70; Riehnet et al., 2007, “Nanochannels for Genomic DNA Analysis: The Long and the Short of It” in Integrated Biochips for DNA Analysis. Springer NewYork, 151-186; Min et al., 2011, “Fast DNA sequencing with a graphene-based nanochannel device.” Nature Nanotechnology 6.3:162-65; U.S. Pat. Nos. 6,673,615; 7,258,838; 7,238,485; 7,189,503; 6,627,067; 6,464,842; and 6,267,872; U.S. Patent Application Publication Nos. 2008/0248561, 2008/0171316, and 2008/0102504; and International Patent Application Publication No. WO 2014/096830, each of which is incorporated herein by reference. Most often, sequence is determined for one single-stranded DNA as it is translated through the nanopore. Both strands of a double-stranded polynucleotide can be sequenced by introducing a hairpin loop at one end of the double-stranded molecule and sequencing the linked sense and antisense strands sequentially (see WO 2013/014451). Sequencing of a double-stranded DNA as it is translated through a nanopore has been suggested (see, Wendell et al., 2009, “Translocation of double-stranded DNA through membrane-adapted phi29 motor protein nanopores” Nature Nanotechnology 4:765-72). In some approaches RNA is sequenced.
Conventional nanopore sequencing is single-pass sequencing, i.e., a single molecule containing one copy of a target sequence is translated through a nanopore one time to generate “single pass sequence information.” Different polynucleotides sharing the same target sequence (e.g., a genomic DNA fragment) may be sequenced by translation through separate nanopores in a multiple pore array to generate multiple reads. The multiple reads can then be used to generate a consensus sequence. A method for moving a polynucleotide in both directions though a nanopore has been proposed, such that the sequence of single molecules might be read in both directions (see Cherf et al., 2012, “Automated Forward and Reverse Ratcheting of DNA in a Nanopore at Five Angstrom Precision” Nat Biotechnol. 30:344-48). However, it is unclear whether any error reduction would result and there appear to be significant barriers to practical implementation of such a system.
Accordingly, improved sequencing methods are needed.